Separation, Purification And Primary Structure Analysis Of Antifreeze Protein Of Xinjiang Cold Water Fish

Antifreeze proteins(AFP), a kind of protein which produced when body resist external cold stress reaction, is widely distributed in animal, plants and microorganisms. Antifreeze protein is a non colligative freezing point, reduce the recrystallization characteristic protein. According to reports, AFP is widely used in food, medical and biological, chemical and other fields owing to its ability of improving the quality of products, improving the ability of cold resistance of crops and improving the survival rate of frozen cells.In this paper, we found AFP existed in skin and flesh when using Esox lucius as raw material for research. Antifreeze protein was separated and purified on these basis and its physical and chemical properties, primary structure was investigated.1 The studies of detection methods of AFPThe commonly used AFP method were compared in this paper. And the differential thermal analyzer method(DSC) was selected for determining the antifreeze activity(AFP THA) detection owing to the advantages including small amount of samples, temperature control accurately,results accurately, etc. Moreover, various factors which affected the result and determination repetition, precision and stability of using DSC method for the determination of THA were studied.Results showed that the DSC method had good stability, repeatability and precision and the RSD value was below 0.4% with relative standard deviation of 3.84%.2 Isolation and purification of antifreeze protein from muscle tissues of Esox luciusIn order to obtain purified antifreeze protein(AFP) from muscle tissues of Esox Lucius, in the present study, AFP was isolated and purified using the single factor experiment and response surface analysis method. Furthermore, four factors, extraction time, extraction temperature, p H value of extraction buffer and the ratios of water to material, was comprehensively detected with thermal hysteresis activity(THA) as the evaluation index. Eventually, evidence from this study indicates that the THA of antifreeze protein from Muscle tissues of Esox Lucius was up to0.0612℃ under the optimized condition that extraction temperature of 5.14℃, sample solutions p H of 8.40, extraction time of 1.88 h and the ratio of 1∶2(g:m L), respectively. In addition, for the sake of verifying the reliability of the model, the verification experiment was implemented. The result revealed that the THA was(0.0594±0.0013)℃, similar to the experiment value. Antifreeze protein of electrophoresis level, the molecular weight of 7.6 Ku, was obtained by further chromatographic purification. Simultaneously, the THA was about(0.052±0.0015)℃.3 Isolation and Purification of Antifreeze Protein from Skin Tissues of Esox LuciusIn order to obtain purified antifreeze protein(AFP) from skin tissues of Esox Lucius, in the present study, AFP was isolated and purified with the help of the single factor experiment and response surface analysis method. Furthermore, four factors, extraction time, extraction temperature, p H value of extraction buffer and the ratios of water to material, wascomprehensively detected with thermal hysteresis activity(THA) as the evaluation index.Eventually, evidence from this study indicates that the THA of antifreeze protein from skin tissues of Esox Lucius was up to 0.072℃ under the optimized condition that extraction temperature of8℃, sample solutions p H of 8.4, extraction time of 2 h and the ratio of 1∶3.8, respectively. In addition, for the sake of verifying the reliability of the model, the verification experiment was implemented. The result revealed that the THA was(0.069+0.0024)℃, similar to the experiment value. Antifreeze protein of electrophoresis level, the molecular weight of 6400, was obtained by further chromatographic purification. Simultaneously, the THA was about(0.052±0.0015)℃.4 Identification of Esox lucius antifreeze proteinThe purified protein was analyzed using HPLC and mass spectrometry. In addition, the target protein was identified by the N-terminal sequencer. The results showed that, the isolated muscle tissues of Esox Lucius antifreeze protein(ELMAFP) was high purity with relative molecular mass of 7.4 KDa and its N-terminal amino acid sequence was LVSLIPIIMPNMVKTVNMTQSMPNMTLNMT. Besides, the skin tissues of Esox Lucius antifreeze protein(ELSAFP) was isolated with high purity. Its relative molecular mass was about6.3 KDa with N-terminal amino acid sequence of MMVFVLLCTQLIPIN. Moreover, the antifreeze protein structure sequence has been found to have similar part from the current reporting through homology analysis and comparison.